The SV40 and polyoma viral replicons provide opportunities to study both the mechanism and the regulation of initiation of eukaryotic DNA synthesis. Far less is understood about polyoma large T antigen and ori- DNA replication than about SV40. However since there are both potentially interesting differences as well as similarities between the two viral replicons, expanding the state of knowledge about polyoma is expected to give additional insight into eukaryotic DNA replication. Three proteins are required for initiating synthesis from the viral replication origin: T antigen, murine DNA polymerase at primase complex (pol alpha), and single-stranded DNA binding protein (RP-A). All are phosphoproteins that are substrates for cyclin dependent protein kinases. We will examine in detail the interactions of T antigen with the origin either alone or with the other two cellular proteins. The regulation by phosphorylation of the three proteins, either separately or functioning together to initiate DNA synthesis, will be analyzed. In a series of related experiments the role of the Rb tumor suppressor protein in inhibiting polyoma ori-DNA synthesis will be characterized. Finally, we will examine how transcriptional activators facilitate the initiation of polyoma ori-DNA synthesis in vitro. Hopefully the planned studies will shed light on the deregulation of cellular DNA synthesis that is a hallmark of tumor cells.